(1) Summary of the Invention
The present invention relates to the disruption of an AbsA gene in a bacterium, particularly a Streptomyces sp. so that there is hyperproduction of an antibiotic naturally produced by the bacterium. Streptomycete antibiotic synthesis in the wild type strain is coupled to morphological differentiation such that antibiotics are produced as a colony sporulates in the wild type strain. Streptomyces coelicolor produces several structurally and genetically distinct antibiotics. It has been found that the S. coelicolor absA locus was defined by UV-induced mutations that globally blocked antibiotic biosynthesis without blocking morphological differentiation. The present invention shows that the absA locus encodes a eubacterial two component sensor-kinase/response regulator system. All four UV mutations lie within a single open reading frame, designated absA1, which codes for the sensor-histidine kinase. A second gene downstream of absA1, absA2, encodes the cognate response regulator. In marked contrast to the antibiotic deficient phenotype of the previously described UV absA mutants, disruption mutations in the absA locus unexpectedly cause premature transcription of the biosynthetic genes for the antibiotics actinorhodin and undecylprodigiosin and precocious hyperproduction of both of these antibiotics. Thus the absA locus encodes a signal transduction mechanism that negatively regulates synthesis of the multiple antibiotics produced by S. coelicolor. This is also true of related Streptomiyces sp.
(2) Description of Related Art
U.S. Pat. No. 4,362,816 to Reusser, U.S. Pat. No. 4,898,828 to Hershberger et al, U.S. Pat. No. 5,118,617 to Ortega et al, U.S. Pat. No. 5,122,595 to Ortega et al, U.S. Pat. No. 5,264,354 to Solenberq, U.S. Pat. No. 5,435,730 to Adams et al, and U.S. Pat. No. 5,474,912 to Sherman et al are of general interest. They describe various related species of Streptomyces.
The widespread use of chemotherapeutic agents has probably been the most significant advance in medicine in this century. The vast variety of compounds available for human health care is due in large part to the biosynthetic versatility of the streptomycetes. These bacteria have provided many thousands of structurally diverse, low molecular weight chemicals that are currently being exploited in both medicine and agriculture. Many streptomycete secondary metabolites have found commercial applications. Some of these are antibacterial drugs, including streptomycin and tetracycline; antiparasitic drugs such as avermectin; fungicidal agents such as polyoxin; antitumor drugs such as adriamycin and immunosuppressive drugs such as rapamycin. The biosynthetic pathways that produce these compounds have received considerable attention over the last few decades.
Advances in understanding the regulation of antibiotic synthesis in Streptomyces have come from the study of antibiotic synthesis in the genetically well characterized strain Streptomyces coelicolor. The four antibiotics produced by S. coelicolor, actinorhodin (Act), undecylprodigiosin (Red), calcium dependent antibiotic (CDA) and methylenomycin (Mmy), are biosynthetically and genetically distinct, genes for the synthesis of each of the antibiotics being encoded in genetically unlinked clusters. All four biosynthetic loci have been genetically well characterized and three biosynthetic gene clusters have been cloned; the act genes; the red genes; and the muny genes. Expression of act biosynthetic genes has been shown to depend on a gene, linked to the biosynthetic genes, designated actII-ORF4 (Fernandez-Moreno, M. A., et al., Cell 66:769-780 (1991); Gramajo, H. C., et al., Mol. Microbiol. 7:837-845 (1993)). Similarly, redD, which is linked to red biosynthetic genes, is required for expression of at least some red genes. ActII-ORF4 and RedD have been termed pathway-specific activators, and numerous antibiotic biosynthetic gene clusters in a variety of streptomycetes require the function of a genetically-linked pathway-specific activator for expression.
In addition to genes whose products specifically regulate the expression of one of the antibiotic gene clusters, a number of loci have been identified which contain genes that globally regulate more than one of the antibiotic biosynthetic clusters. Mutations in some of these, the bld loci, pleitropically block the synthesis of all four antibiotics as well as the production of sporulating aerial hyphae (Champness, W. C., J. Bacteriol. 170:1168-1174 (1988)). Other genes including abaA (Fernandez-Moreno, M. A., et al., J. Bacteriol. 174:2958-2967 (1992)), afsQ1-Q2 (Ishizuka, H., et al., J. Bacteriol. 174:7585-7594 (1992)) and afsR-K-R2 (Stein, D., et al., J. Bacteriol. 171:2258-2261 (1989)), play a role in the regulation of two or three of the antibiotics.
Mutations in two loci, absA (Adamidis, T., et al., J. Bacteriol. 172:2962-2969 (1990)) and absB (Adamidis, T., et al., J. Bacteriol. 174:4622-4628 (1992)) are known to block the synthesis of all four antibiotics, while having no pleiotropic effects upon morphological development. In addition suppressors of absA mutations (sab) exist which restore antibiotic synthesis to normal or even greater than normal levels. Thus the prior art has presumed that modification of the absA gene interrupts antibiotic production.